Identification of a 16SrIX-B phytoplasma strain associated with Daphne mucronata phyllody in Iran

During surveys from 2017 to 2018, a phyllody disease was observed in Daphne mucronata plants located in Lordegan and Ahmadliveh areas in Chaharmahal and Bakhtiari province (west of Iran). The main symptoms were yellowing, little leaf, phyllody and flower sterility. Total DNA was extracted from midrib tissue of fresh leaves from 30 symptomatic and five asymptomatic plants. DNA samples were subjected to nested PCR, using primer pairs P1/P7 and R16F2n/R16R2. PCR amplicons of 1.8 and 1.25 kb, respectively, were obtained from all samples from symptomatic plants whilst no amplification was observed in samples from asymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using MseI, HhaI, AluI, HaeIII, RsaI, HpaII, TaqI, KpnI, ThaI and BfaI enzymes showed identical RFLP patterns in all the samples that were also identical to the profiles of the 16SrIX-B phytoplasma subgroup. Sanger sequencing of five randomly selected amplicons confirmed that they were 100% identical to each other and had >99% identity with ‘Candidatus Phytoplasma phoenicium’-related strains as also confirmed by the clustering in phylogenetic analysis. This is the first report of 16SrIX-B phytoplasma presence in a non-Prunus species, D. mucronata, also highlighting its possible role as reservoir species for this phytoplasma that is severely affecting stone fruits in the Middle East regions.